Peptide nucleic acid (PNA) is a DNA analogue linked by peptide bonds, not by phosphate bonds, and was first reported in 1991 [Nielsen P E, Egholm M, Berg R H, Buchardt O, “Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide”, Science 1991, Vol. 254: pp. 1497-1500] (FIG. 1). PNA is synthesized chemically and is not known to occur naturally. PNA hybridizes to a naturally occurring nucleic acid with a complementary base sequence to form a double strand. Given the same number of nucleic acid bases, a PNA/DNA double strand is more stable than a DNA/DNA double strand, and a PNA/RNA double strand is more stable than a DNA/RNA double strand. The most frequently used backbone of PNA is repeating N-(2-aminoethyl)glycine units linked by amide bonds. The PNA's backbone is electrically neutral, whereas naturally occurring nucleic acids are negatively charged. The four nucleobases of PNA occupy similar spaces to those of DNA, and the distance between the nucleobases is almost identical to that in the naturally occurring nucleic acids. PNA is not only chemically more stable than naturally occurring nucleic acids but also biologically more stable since it is not degraded by nucleases or proteases. Also, because PNA is electrically neutral, the stability of the PNA/DNA and PNA/RNA double strands is not affected by salt concentration. For these reasons, PNA better recognizes complementary base sequences than naturally occurring nucleic acids and is utilized for diagnosis or other biological or medical applications.
In general, when a sequence of nucleobase is recognized or detected in a homogeneous solution using a probe with a known base sequence, only one sequence can be recognized at a time, and it is difficult to detect several sequences at once using fluorescent dyes of different colors. In contrast, by immobilizing a great number of probes on a solid surface, a number of specific sequences of nucleobase may be detected at once. A DNA microarray on which hundreds of thousands of probes are two-dimensionally arranged is commercially available. Also, a PNA microarray or a PNA chip using a PNA probe instead of a DNA probe is known [Brandt O, Hoheisel J D, “Peptide nucleic acids on microarrays and other biosensors” Trends Biotechnology 2004, Vol. 22, pp. 617-622]. A technique of immobilizing PNA probes on the surface of microbeads (or microspheres) of several μm size to carry out detection is also known [Rockenbauer E, Petersen K H, Vogel U, Bolund L, Kølvraa S, Nielsen K V, Nexø B A, “SNP genotyping using microsphere-linked PNA and flow cytometric detection” Cytometry Part A 2005, Vol. 64A, pp. 80-86]. Although a technique of identifying hybridization of a probe to a complementary sequence of nucleobase using fluorescence is widely used, a technique of detecting a sequence of nucleobase electrically using a field-effect transistor using PNA immobilized on a silicon semiconductor or silicon nanowire is also known [F. Uslu et al. “Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device”, Biosensors and Bioelectronics 2004, Vol. 19 pp. 1723-1731; J. Hahm and C. M. Lieber, “Direct ultrasensitive electrical detection of DNA and DNA sequence variations using nanowire nanosensors”, Nano Letters 2004, Vol. 4, pp. 51-54]. Also, an apparatus for detecting a sequence of nucleobase based on impedance change is reported [A. Macanovic et al. “Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer”, Nucleic Acids Research 2004, Vol. 32, p. 20].
Since the mass of a probe changes before and after hybridization to a target nucleic acid, a sequence of nucleobase may be detected based on the mechanical change resulting therefrom. Also, detection can be made based on the fact that the vibration frequency of a microcantilever or a surface acoustic wave (SAW) sensor changes before and after binding to DNA or RNA. A microcantilever and a SAW sensor using PNA are reported [S. Manalis and T. Burg, U.S. Pat. No. 7,282,329 “Suspended microchannel detectors”; P. Warthoe and S. Iben, US Patent Application Publication No. 2004/0072208 A1 “Surface acoustic wave sensors and method for detecting target analytes”].
Such apparatuses or methods of detecting base sequences using a plurality of PNA probes require immobilization of the PNA probes on solid surface. For the immobilization, stable chemical covalent bonding is more frequently employed than physical bonding. In general, immobilization using a covalent bonding such as aldehyde-amine bonding, carboxylic acid-amine bonding or epoxide-amine bonding is widely employed for immobilization of a biochip such as a PNA chip, a DNA chip, a protein chip, or the like [M. Schena, Microarray analysis, A John Wiley & Sons, Inc., Publication, pp. 95-120]. In order to immobilize PNA on glass surface, the glass surface is often subject to silylation by an organosilane substance having an aldehyde, amine or epoxy group so that the functional group is exposed on the glass surface. Then, the N-terminal amine group of PNA is reacted with the exposed functional group to form a covalent bonding.
When a probe is immobilized on the solid surface, if the probe is too close to the support, steric hindrance may occur during hybridization of the probe to the target gene. A spacer is interposed between the probe and the solid support to solve this problem. A nucleotide spacer linked by phosphate bonding and an amino acid spacer with a relatively short chain may be used for this purpose. The spacer greatly influences the interaction of the probe with the target substance depending on its length, charge, hydrophobicity, solvation property or the like.
In a DNA chip, a nucleotide spacer linked by phosphate bonding is mainly used to improve sensitivity and specificity of target gene detection [Magdalena Gabig, Acta Bio. Polonica, 2001, 48, 615]. However, the nucleotide spacer linked by phosphate bonding is problematic in that it is not applicable to a PNA having a backbone linked by amide bonding since it lacks amine, carboxylic acid or ester residues that form amide bonding. In addition, the phosphate anion decreases the efficiency of hybridization [W. Pils and R. Micura, Nucleic Acids Research, 2000, 28, 1859.; U.S. Pat. No. 7,205,104]. Further, a spacer having positive or negative charge is known to have decreased efficiency of hybridization as compared to a neutral spacer [M. S. Shchepinov, Nucleic Acids Research, 1997, 25, 1155]. For this reason, neutral amino acid derivatives with linear structure such as 8-amino-3,6-dioxaoctanoic acid that can form amide binding with PNA were introduced. By synthesizing a probe by polymerizing several 8-amino-3,6-dioxaoctanoic acids with a PNA oligomer and immobilizing it on a support, thereby ensuring a space between the support and the PNA oligomer, the efficiency of hybridization may be improved.
However, because 8-amino-3,6-dioxaoctanoic acid has a short length, polymerization of 8-amino-3,6-dioxaoctanoic acid to the PNA oligomer has to be repeated for 4-5 times or more to ensure a sufficient distance between the support and PNA. Thus, there is a need for using a longer spacer so that a sufficient distance between the PNA oligomer and the support can be ensured without having to perform the polymerization several times.